Yersinia enterocolitica, an enteric pathogen of humans, is able to cross the epithelial cell boundary in the small intestine and gain access to the cells of the reticuloendothelial system below. A large plasmid common to all species of Yersinia able to cause disease in humans has been demonstrated to be required for virulence. A number of genes encoded on this plasmid have been identified. Although the plasmid has been demonstrated to be necessary for virulence, it is not sufficient. The chromosomal background of the strain also has an important role in the pathogenesis of this organism. An in vitro model of bacterial entry into mammalian cells has been developed to mimic the entry of bacteria into both epithelial cells and reticuloendothelial cells in vivo. Two chromosomal genes from Y. enterocolitica have been identified as having a role in the in vitro entry process; both genes confer an invasive phenotype when expressed in noninvasive laboratory strains of E. coli. To demonstrate that these genes, called inv and ail, do have a role in pathogenesis by Y. enterocolitica, strains containing mutations in these genes will be studied in an animal model for yersinial disease. These experiments will also serve to demonstrate whether this in vitro model is a valid model for in vivo events. To determine where in the pathogenesis process these two genes act, the regulation of these genes by environmental factors known to vary during the course of infection will be examined. The production of transcripts homologous to these two genes will be examined in bacteria grown under a variety of environmental conditions by Northern analysis and by measuring production of enzyme activity from transcriptional fusions to reporter genes. By defining the point in infection that the inv and ail genes are active, these studies will further demonstrate the role of these genes in pathogenesis. These studies will also allow identification of genes that regulate the inv and ail genes and to determine if these regulatory genes have an effect upon other virulence factors of this organism. The third objective of this proposal is to carry out a detailed analysis of the function of the product of the ail gene of Y. enterocolitica. The expression of the ail gene in certain nonpathogenic strains of Y. enterocolitica is not sufficient to confer the invasive or adherent phenotypes to these strains. The studies proposed here will allow us to better understand the requirements for ail-mediated invasion and adherence. The additional bacterial factors that are involved in these processes will be identified by cloning into and mutagenizing the strains that express ail and selecting for invasive/adherent cells. Completion of these studies will identify and help to understand the function of these factors in pathogenesis and bacterial/host interactions.